CN107502596A - Express T cell and its application of the specificity TCRs of NY ESO 1 - Google Patents
Express T cell and its application of the specificity TCRs of NY ESO 1 Download PDFInfo
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Abstract
The invention provides T cell, and it includes coding specific recognition NY ESO 1 TCR nucleic acid and expresses the TCR of the specific recognition NY ESO 1.Present invention also offers the TCR for expressing the specific recognition NY ESO 1, and IL 2 T cell is expressed simultaneously.Present invention also offers the recombinant expression carrier of coding specific recognition NY ESO 1 TCR nucleic acid.Present invention also offers purposes of the T cell of the present invention in the medicine for preparing prevention or treating cancer.
Description
Technical field
The present invention relates to immunology and field of medicaments.Specifically, the invention provides contain specific recognition NY-ESO-1
φt cell receptor and express the T cell of the TCR and cell factor simultaneously.Present invention also offers the T cell prevention or
Purposes in treating cancer or the medicine of preparation prevention or treating cancer.
Background technology
In recent years, immunotherapy of tumors is quickly grown, but whole structure is not still good enough.To find out its cause, it may lead to tumour
It is relevant to cross various mechanism generation immunologic escapes.As tumor cells expression with normal tissue cell identical antigen is escaped body and exempted from
The identification of epidemic disease system；The function of immunocompetent cell is suppressed by secretory immune inhibiting factor such as TGF-β and IL-10 etc.；It is logical
Downward TNF-α acceptor, Fas acceptors etc. are crossed to resist cell factor and cytotoxic T lymphocyte (cytotoxic
Lymphocyte, CTL) GVT；Prevent antigen from effectively offering by losing tumour antigen downward HLA.Tumour
Infiltrating T cells (tumor-infiltrating lymphocyte, TIL) are although adoptive immunotherapy takes in Partial tumors
It must succeed, but not got a desired effect in most of tumours.Its main cause includes：TIL is difficult to obtain, these
Functional activity declines after T cell amplification in vitro, reaches the T cell lazy weight of tumor tissues and in tumor tissues microenvironment
Lose immunocompetence etc..Therefore, researcher has attempted solve this problem using genetic modification T cell, it is such as thin for modifying T
The gene of born of the same parents has CAR (chimeric antigen receptor, mosaic type antigen receptor), promotes the thin of immune cell propagation
Intracellular cytokine such as IL-2, IL-15 etc. have obtained the effect of preferable, but because the problems such as missing the target property, security significantly limit
Their applications clinically.
T cell adoptive transfer can strengthen the elimination to tumour cell of immune-mediated, be obtain in recent years compared with
Specific, the avirulent new treatment for cancer of height concern.In T cell adoptive transfer, φt cell receptor (TCR) gene transfer is made
For a kind of immunotherapy method quickly grown, can produce in vitro largely has known antigens specificity and function affine
The T cell of property, the adoptive cellular immunotherapy applied to malignant tumour.Tcr gene engineering T cell generation screen first and
Clonal tumours specificity TCR gene, then with transfecting T cells assign its antigentic specificity, it is genetically engineered anti-so as to obtain
Former specific T-cells, most tcr gene transfecting T cells feed back patient's body at last, rebuild the T cell for antigen positive tumour
Immune response.
So far, the TCR candidates of therapeutic action or a small amount of are suitable for use in.In addition, the T of the TCR transductions of these identifications is thin
The problems such as T cell activation signal transduction is weak and time-to-live in vivo is short still be present in born of the same parents.
NY-ESO-1 belongs to Cancer Testis Antigens (CTA) family, and it does not express MHC tumour cell and testis and placenta
Reproduction cell in express (Chen, PNAS USA, 1997,94 (5):1914-1918).It has been found that NY-ESO-1 is in a variety of people
Expressed in cancer, including melanoma, breast cancer, lung cancer, prostate cancer, thyroid cancer, oophoroma, osteosarcoma and synovial cell's meat
Knurl.
Osteosarcoma is a kind of malignant tumour originating from bone mesenchymal tissue, accounts for the 20% of primary malignant bone tumor, about 70%-
80% morbidity's age, annual morbidity was about the people of (1-3)/1,000,000 10-25 year.Osteosarcoma grade malignancy is high, has in early days
Lung metastases may occur, the patient that there are about 10-20% has found there is transfer stove, poor prognosis in diagnosis.At present, although people taste
New chemotherapeutics or drug combination on probation, the survival rate of patient no longer improve.
Interleukin 2 (interleukin 2, IL-2) is a kind of cell factor with extensive immunologic competence, main
To be produced by CD4+ and CD8+T cells.It plays very important effect in immune system：Promote all hypotype T cells
Propagation and generation cell factor, extend its life cycle；Promote NK cytotoxic activities；Activated mononuclear/macrophage, and strengthen it and kill
Tumor activity etc..IL-2 also achieves certain immune effect as immunotherapeutic agent and immunologic adjuvant application.IL-2's is antitumor
Effect removes and LAK, TIL have outside the Pass, also relevant with the generation that it induces NO, but because IL-2 is expensive, Half-life in vivo is short, big
Amount injection adverse reaction weight, so as to limit its application.
This area also needs to obtain new antigen specific T CR, and obtains the T cell for expressing these TCR, while can lead to
Other immune means are crossed, increase the activity of these T cells, to be more effectively carried out antigen specific immunotherapy.
The content of the invention
The invention provides a kind of new specific recognition NY-ESO-1 φt cell receptor, i.e. TCR, and itself or its
Combine the application expressed in T cell with cell factor, particularly interleukin 2 (IL-2).The invention provides expression
TCR and IL-2 T cell described in the TCR or co expression, the T cell is with NY-ESO-1 antigentic specificities and with more
High multiplication capacity.Present invention also offers include the TCR or its coded sequence and energy Efficient Conversion T cell with IL-2
Lentiviral.Present invention also offers the method using the T cell treating cancer for expressing the TCR or prepare treatment
The purposes of the medicine of cancer.
The invention provides a kind of T cell, and it includes coding specific recognition NY-ESO-1 TCR nucleic acid and expresses institute
Specific recognition NY-ESO-1 TCR is stated, the TCR includes α chains and β chains.In the one aspect of the present invention, the coding
Specific recognition NY-ESO-1 TCR nucleic acid includes the first nucleic acid fragment for encoding the α chains of the TCR.Preferably, described
It is SEQ ID NO that one nucleic acid fragment, which has sequence,：1 nucleotide sequence.In the wherein another aspect of the present invention, the nucleic acid
Also include the second nucleic acid fragment for encoding the β chains of the TCR.Preferably, it is SEQ ID that second nucleic acid fragment, which has sequence,
NO：2 nucleotide sequence.In the wherein another aspect of the present invention, the TCR of coding specific recognition NY-ESO-1 core
Acid further comprises the joint between first nucleic acid fragment and the second nucleic acid fragment.Preferably, the joint is coding T2A
Nucleotide sequence, for example, the nucleotides sequence of the joint is classified as AGGGCTAAACGCTCCGGGTCTGGAGCAACAAACTTCTCT
CTGCTGAAGCAGGCTGGCGATGTGGAGGAAAATCCTGGGCCA(SEQ ID NO：4).
NY-ESO-1 is the antigen for belonging to Cancer Testis Antigens family.In the present invention, NY-ESO-1 is primarily referred to as people NY-
ESO-1, its protein sequence such as GenBank:CAA05908.1 is defined.The gene for encoding NY-ESO-1 is CTAG1B (cancer/
Testis antigen 1B), its nucleotide sequence such as Gene ID:1485 are defined.
In the wherein another aspect of the present invention, foregoing T cell can also express interleukin 2, i.e. IL- comprising coding
2 nucleic acid simultaneously expresses interleukin 2.Preferably, it is SEQ ID NO that the nucleic acid of the encoding Interleukin 2, which has sequence,：
3 nucleotide sequence.In the wherein another aspect of the present invention, the nucleic acid of the encoding Interleukin 2 is by encoding T2A's
Nucleotide sequence is connected with the TCR of coding specific recognition NY-ESO-1 nucleic acid.The nucleotides of the nucleic acid of the coding T2A
Sequence is, for example, AGGGCTAAACGCTCCGGGTCTGGAGCAACAAACTTCTCTCTGCTGAAGCAGGCTGG CGATGTGGAGG
AAAATCCTGGGCCA(SEQ ID NO：4).
T cell provided by the invention being capable of specific recognition expression or the cell in cell surface presentation NY-ESO-1.This
The T cell of invention can be CD8+T cells and CD4+T cells.In one aspect of the invention, T cell of the invention is CD8+T
Cell.T cell provided by the invention presents NY-ESO-1 cell, including cancer cell to expression or in cell surface, kills
Overstrain, ruptures cancer cell and dead, and can turn into memory cell, will be rapider when running into same antigen stimulation again
Ground Proliferation, Differentiation.
Present invention also offers the recombinant expression carrier of the nucleic acid of the TCR comprising coding specific recognition NY-ESO-1.
The one aspect of the present invention, the TCR of coding specific recognition NY-ESO-1 nucleic acid include the α for encoding the TCR
First nucleic acid fragment of chain.Preferably, it is SEQ ID NO that first nucleic acid fragment, which has sequence,：1 nucleotide sequence.
The wherein another aspect of the present invention, the nucleic acid include the second nucleic acid fragment for encoding the β chains of the TCR.Preferably, it is described
It is SEQ ID NO that second nucleic acid fragment, which has sequence,：2 nucleotide sequence.In the wherein another aspect of the present invention, the volume
Code specific recognition NY-ESO-1 TCR nucleic acid further comprises connecting first nucleic acid fragment and the second nucleic acid fragment
Joint.Preferably, the joint is encodes T2A nucleotide sequence, for example, the nucleotides sequence of the joint is classified as SEQ ID NO：
4 nucleotide sequence.
In the wherein another aspect of the present invention, the recombinant expression carrier can also express interleukins comprising coding
2 nucleic acid.Preferably, it is SEQ ID NO that the nucleic acid of the encoding Interleukin 2, which has sequence,：3 nucleotide sequence.
The wherein another aspect of the present invention, the nucleic acid of the encoding Interleukin 2 are known by T2A sequences and the coding specificity
Other NY-ESO-1 TCR nucleic acid connection, the T2A sequences are, for example, SEQ ID NO：4 nucleotide sequence.
In the one aspect of the present invention, recombinant expression carrier of the invention is plasmid.In the wherein another of the present invention
Individual aspect, recombinant expression carrier of the invention are the plasmids for packaging virus carrier, are e.g. used to pack baculoviral table
Up to carrier, adenovirus vector, retroviral vector, the plasmid of herpesvirus vector or slow virus carrier etc..
In the one aspect of the present invention, recombinant expression carrier of the invention is the load of the expressing protein in mammal
Body.In the wherein another aspect of the present invention, the recombinant expression carrier is viral vector, such as rhabdovirus expression vector,
Adenovirus vector, retroviral vector, herpesvirus vector or slow virus carrier etc..Preferably, recombination expression of the invention
Carrier is slow virus carrier.In viral vector, exogenous DNA Insert Fragment is connected in viral genome, and can infect target
Cell (such as T cell) and bring exogenous DNA Insert Fragment into target cell/be incorporated into target cell genome, and express
Its albumen encoded.
The present invention recombinant expression carrier except the TCR including above-mentioned coding specific recognition NY-ESO-1 nucleic acid and/or
The nucleic acid of encoding Interleukin 2, in addition to other sequences can be included, such as：The sequence that regulation carrier replicates in host cell
(such as：Replication orgin) and selected marker.
In one aspect of the invention, there is provided the medicine group of T cell or recombinant expression carrier containing the invention described above
Compound.In one aspect of the invention, there is provided the T cell of the invention described above or recombinant expression carrier for prepare treatment or
Purposes in the medicine of pre- anti-cancer.The recombinant expression carrier of the present invention can will encode specific recognition NY-ESO-1 TCR
And/or IL-2 nucleic acid is expressed in T cell.The T cell of the present invention has specific recognition NY-ESO-1, passes through cytotoxicity
Effect and the generation of lymphokine play antitumor action.Therefore, T cell of the invention can be used for treating or preventing cancer, i.e.,
Can be for the active component for the pharmaceutical composition for treating or preventing tumour.
The method of the administration of the aforementioned pharmaceutical compositions of the present invention includes intracutaneous, subcutaneous, intramuscular, intravenous administration etc..
The method of the administration of the pharmaceutical composition of the present invention includes intravenous apply.The reagent of T cell containing the present invention can be returned
Inside patient, therefore tumour can effectively killed by the patient's body that T cell of the present invention identifies and has reaction, as a result can treated
Or pre- preventing tumor.
The pharmaceutical composition of the invention described above can be used for prevention or treating cancer.The cancer includes leukemia, solid tumor etc.,
More particularly, including melanoma, breast cancer, lung cancer, prostate cancer, thyroid cancer, oophoroma, osteosarcoma and synovial cell's meat
Knurl.Preferably, the pharmaceutical composition of T cell or recombinant expression carrier comprising the present invention can be used for preventing or treat osteosarcoma.
Herein, albumen symbol does not have to italic, and all Caps；Gene symbol uses italic.Such as NY-ESO-1 tables
Show antigen protein, the gene for encoding the albumen is written as CTAG1B.But gene symbol herein is also without using italic sometimes.
Herein, term " nucleic acid or nucleic acid fragment of coded polypeptide or protein " includes containing more peptide or proteins
The nucleotides of matter coded sequence.Except the polypeptide or the nucleotides of protein coding sequence, the nucleic acid or nucleic acid fragment may be used also
Including other single continuums or discontinuity zone (for example, polynucleotides are integrated bacteriophage, integration insetion sequence, integration
Carrier sequence, integrate transposon sequence interrupt or be interrupted due to rna editing or genomic DNA reorganization) albumen or non-egg
The nucleotides of white coded sequence, such as the sequence of the integration for assisting Insert Fragment, the sequence of expression regulation, encoded signal peptide
Sequence etc..
Brief description of the drawings
Fig. 1 plasmid pGreen puro-TCR (NY-ESO-1) provided by the invention and pGreenpuro-TCR (NY-ESO-
1) structural representation of-IL2 Insert Fragment.
Fig. 2 is the volume of the Insert Fragment of recombinant expression carrier pGreen puro-TCR (NY-ESO-1) provided by the invention
Code base sequence.
Fig. 3 is recombinant expression carrier pGreen puro-TCR (NY-ESO-1)-IL2 provided by the invention Insert Fragment
Encoding base sequences.
Fig. 4 is the flow cytometer inspection of the T Expressions In Lymphocytes NY-ESO-1 specificity TCRs of the plasmid transfection of the present invention
Survey result figure.
Fig. 5 is the T lymphocyte antitumous effects of the plasmid transfection of the present invention.
Embodiment
The substantive content and beneficial effect of the present invention is further illustrated below in conjunction with embodiment, the embodiment is only used for
The bright present invention rather than limitation of the present invention.
The recombinant expression carrier pGreen puro-TCR (NY-ESO-1) of embodiment 1 structure
Construct recombinant expression carrier pGreen puro-TCR (NY-ESO-1).The structure of the Insert Fragment of carrier such as Fig. 1
It is shown.PGreen puro-TCR (NY-ESO-1) Insert Fragment includes the first nucleic acid fragment and coding of coding TCR α chains
Second nucleic acid fragment of TCR β chains；First nucleic acid fragment and the second nucleic acid fragment are connected by T2A sequences.
Building process includes：
Composite coding NY-ESO-1TCR α-T2A-NY-ESO-1TCR β (being referred to as TCR (NY-ESO-1) sequence) nucleic acid piece
Section, its first nucleic acid fragment (base sequence is as shown in SEQ ID No.1) as the α chains including encoding NY-ESO-1-TCR, bag
Include the second nucleotide sequence (base sequence is as shown in SEQ ID No.2) of coding NY-ESO-1-TCR β chains and by described the
The T2A sequences that one nucleic acid fragment and the second nucleic acid fragment are together in series
(AGGGCTAAACGCTCCGGGTCTGGAGCAACAAACTTCTCTCTGCTGAAGCAGGCTGGCGATGTGGAGGAAAATCCTG
GGCCA) form.The nucleic acid fragment of TCR (NY-ESO-1) sequence is as shown in Figure 2.The sequence of its medium and small sizes font is coding
The nucleic acid of T2A sequences；It is the first nucleic acid fragment before the sequence of small size font；It is the second nucleic acid fragment after the sequence of small size font.
EcoR I and BamHI restriction enzymes position is added at the nucleic acid fragment both ends of the TCR (NY-ESO-1) sequence
Point sequence.The nucleic acid fragment and slow virus core carrier pGreen puro restriction enzyme EcoR I and BamH I are entered
Row double digestion, after digestion products gel extraction, with the 16 DEG C of connections overnight of T4 ligases.Escherichia coli HB2151 senses are converted after connection
By state, spread plate, next day picking monoclonal double digestion and sequencing identification, recombinant expression carrier pGreen puro-TCR are obtained
(NY-ESO-1)。
Embodiment 2 recombinant expression carrier pGreen puro-TCR (NY-ESO-1)-IL2 structure
PGreen puro-TCR (NY-ESO-1)-IL2 Insert Fragment includes the first nucleic acid fragment of coding TCR α chains
With the second nucleic acid fragment of coding TCR β chains, and the 3rd nucleic acid fragment of encoding Interleukin 2；First nucleic acid fragment and
Second nucleic acid fragment is connected by T2A sequences；Second nucleic acid fragment and the 3rd nucleic acid fragment are connected by T2A sequences.
Building process includes：
Composite coding NY-ESO-1TCR α-T2A-NY-ESO-1TCR β and IL-2 nucleic acid fragment (are referred to as TCR (NY-ESO-
1)-IL2 sequences), its by including encode NY-ESO-1-TCR α chains the first nucleic acid fragment (base sequence such as SEQ ID No.1
It is shown) including coding NY-ESO-1TCR β chains second nucleotide sequence (base sequence is as shown in SEQ ID No.2) including
Encode IL-2 the 3rd nucleic acid fragment (base sequence is as shown in SEQ ID No.3) and in first nucleic acid fragment and the
Two T2A sequences between two nucleic acid fragments, between the second nucleic acid fragment and the 3rd nucleic acid fragment (are used for three sequences
It is together in series) composition.The nucleic acid fragment of the Insert Fragment of TCR (the NY-ESO-1)-IL2 sequences is as shown in Figure 3.It is wherein previous
The sequence of individual small size font is the first nucleic acid fragment before its sequence, is the second nucleic acid after its sequence to encode the nucleic acid of T2A sequences
Fragment；The sequence of the latter trumpet font is the 3rd nucleic acid fragment after its sequence to encode the nucleic acid of T2A sequences.
EcoR I and BamH I restriction enzymes are added at the nucleic acid fragment both ends of the TCR (NY-ESO-1)-IL2 sequences
Enzyme site sequence.By the nucleic acid fragment and slow virus core carrier pGreen puro restriction enzyme EcoR I and BamH
I carries out double digestion, after digestion products gel extraction, with the 16 DEG C of connections overnight of T4 ligases.Escherichia coli are converted after connection
HB2151 competence, spread plate, next day picking monoclonal double digestion and sequencing identification, obtain recombinant expression carrier pGreen
puro-TCR(NY-ESO-1)-IL2。
The packaging of the slow virus of embodiment 3
By obtained recombinant expression carrier pGreen puro-TCR (NY-ESO-1) and pGreen puro-TCR (NY-
ESO-1)-IL2 passes through with slow virus skeleton plasmid pMDLg PRRE, pRSV-Rev and pMD2.G (Addgene) equivalent respectively
The liposomes of Lipofectamine 2000 (Life, Invitrogen, article No. 11668-019) transfection enters Hek293T cells
The packaging of slow virus is carried out in (ATCC, article number CRL-3216).
Specification according to slow virus conversion reagent supplier is operated.Specific steps include：With containing 10%FBS's
1640 medium culture 293T cells；Then by 293T cells with 3x105/cm2Density reach and trained in diameter 15cm culture dish
20h is supported, ensures that cell confluency degree is 80-90% during transfection；Liquid is changed with 1640 culture mediums without serum, it is standby；Take two EP
Pipe, 1ml 1640 is added in EP pipes respectively, by slow virus carrier pGreen puro-TCR (NY-ESO-1) or pGreen
Puro-TCR (NY-ESO-1)-IL2 are with pMDLg PRRE, pRSV-Rev and pMD2.G plasmids according to mol ratio 1：1：1：1 in it
In mix in an EP pipe, 150ul Lipo2000 are added in another EP pipe, mixes and places 5min, lipo2000 will be mixed with
1640 culture mediums add the EP pipes containing plasmid in, mix, room temperature place 20min, then mixed liquor is added dropwise above-mentioned
In the Tissue Culture Dish of preparation, continue after cultivating 4h, nutrient solution is changed with 1640 culture mediums containing 10%FBS.It is glimmering to be GFP simultaneously
Light control group, sees transfection efficiency, and virus is packed successfully.Continue collection cell conditioned medium after cultivating 48h and be used for viral purification.
The purifying of the slow virus of embodiment 4
Viral supernatants are collected in 50ml super filter tubes with 0.22 μm of filtering membrane filtration, filtrate, 3000g/min centrifugations
45min；Remaining concentrate is transferred to EP pipes, -80 DEG C is put and saves backup.
Respectively obtain Insert Fragment and pGreenpuro-TCR (NY- with pGreen puro-TCR (NY-ESO-1)
ESO-1) the slow virus carrier of-IL2 Insert Fragment.
The separation of embodiment 5CD8+T cells
A：Whole blood of the collection from volunteer's (signing informed consent form), pour into 50ml centrifuge tubes, room temperature centrifugation 700g
20 minutes (common centrifugation).
B：Above-mentioned centrifugation bottom cell component is taken, adds DPBS to 50ml.
C：Take 25ml aforesaid liquids to be added to 20ml human lymphocyte separating liquids respectively, centrifuge tube room temperature is centrifuged into 800g 15
Minute.
D：Tunica albuginea confluent monolayer cells are taken, DPBS is added, complements to 50ml.
E：Centrifuge 600g 10 minutes, abandon supernatant, obtain PBMC.
F：CD8+T cells are sorted using CD8+T cell magnetic bead sortings kit.
The slow virus carrier of embodiment 6 infects T lymphocytes
With the complete medium culture CD8+T cells of RPMI 1640 containing 10% hyclone, AntiCD3 McAb list is added within first day
Clonal antibody activates；The slow virus being prepared according to embodiment 4 for adding 150MOI (carries pGreen puro- within 3rd day
The slow virus of TCR (NY-ESO-1) Insert Fragment or pGreen puro-TCR (NY-ESO-1)-IL2 Insert Fragment), not
The T lymphocytes of infection are as blank control；Culture medium is replaced by the RPMI containing 50IU/ml recombinant human il-2s after 16h
1640 complete mediums, continue culture 10-20 days, then observe T lymphocyte growth situations.
Observation display：
Cell can form typical proliferating clones group after virus infection.
Expression of the specific recognition NY-ESO-1 of embodiment 7 TCR in T cell
It will cultivate to the T cell of the virus infection of 14 days, 600g centrifugation 10min, abandon most supernatant to collect cell；PBS
Cell is resuspended in solution, and is 1x 10 by cell adjustment density7Individual/ml；100 μ l cells are placed in EP pipes, add 10 μ l FITC
The NY-ESO-1 pentamers (SLLMWITQC) of mark mouse anti human CD8 monoclonal antibodies and 10 μ l PE marks (Proimmune,
Code：F049-2A-G, USA)；4 DEG C of incubation 30min, PBS solution are washed 2 times, and flow cytometer detects TCR correct table
Reach.
Fig. 4 is the CD8+T cell tables of the slow-virus infection of the Insert Fragment with pGreen puro-TCR (NY-ESO-1)
Up to NY-ESO-1 specificity TCR flow cytometer testing result figures.Flow cytometer testing result shows, with pGreen
In the CD8+T cells of the slow-virus infection of puro-TCR (NY-ESO-1) Insert Fragment, the expression of NY-ESO-1 specificity TCRs
For 23.81%.
To be not inserted into the carrier pGreen puro of fragment and skeleton plasmid pMDLg PRRE, pRSV-Rev and pMD2.G
The T cell for the unloaded precursor virus infection that transfection Hek293T cells obtain is as control.The NY-ESO-1 of the T cell of control is special
Property TCR expression rate for 0.46%).
In another experiment, flow cytomery result shows, with pGreen puro-TCR (NY-ESO-1)-IL2
The CD8+T cells of slow-virus infection of Insert Fragment also express NY-ESO-1 specificity TCRs.
Embodiment 8 expresses TCR (NY-ESO-1) and TCR (NY-ESO-1)-IL2 T lymphocytes antitumous effect checking
Using the stable human osteosarcoma cell system Saos-2 (ATCC#HTB-85) for expressing NY-ESO-1 as target cell, respectively
The T infected with slow virus carrier pGreen puro-TCR (NY-ESO-1) and pGreen puro-TCR (NY-ESO-1)-IL2 is thin
Effector cell is made in born of the same parents, by target cell according to density 1x 105Individual/ml is inoculated with 96 orifice plates, per the μ l of hole 100, according to 5:1,10:1,
20:Effector cell is added target cell by 1 effect target ratio, is placed in 5%CO2, 37 DEG C of incubator culture 4h, cell is detected using WST-1
Vigor, killing-efficiency is calculated as follows：
Fragmentation effect (proliferation inhibition rate)=[1- (experimental group OD values-effector cell OD values)/target cell OD values] ×
100%.
As a result it is as shown in Figure 5：
It is provided by the invention only to express NY-ESO-1TCR and express NY-ESO-1TCR and cell factor IL-2 T simultaneously
Lymphocyte all has lethal effect to NY-ESO-1 positive tumor cells.
In addition, the T lymphocytes for expressing NY-ESO-1TCR and cell factor IL-2 simultaneously more only express NY-ESO-1TCR
And the T lymphocytes for not expressing IL-2 have the lethal effect significantly increased to NY-ESO-1 positive tumor cells.
The above is the explanation carried out to the present invention, it is impossible to is regarded as the limitation carried out to the present invention.Unless refer in addition
Go out, practice of the invention will using organic chemistry, polymer chemistry, biotechnology etc. routine techniques, it is clear that except being stated upper
Outside being particularly described in bright and embodiment, the present invention can also be realized otherwise.Other aspects within the scope of the present invention
Will be apparent to those skilled in the art in the invention with improving.According to the teachings of the present invention, many changes and change are
Feasible, therefore it is within the scope of the present invention.
DEG C as without particularly showing, the unit " degree " of herein presented temperature refers to degree Celsius, i.e.,.
Sequence table
<110>Beike Biological Sci-Tech Co., Ltd., Shenzhen
<120>Express T cell and its application of NY-ESO-1 specificity TCRs
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 819
<212> DNA
<213>Artificial sequence ()
<400> 1
atgactctga agatggatag ctccccaggc ttcgtggccg tgatcctgct catcctggga 60
cggacccacg gcgactccgt gacccagaca gaggggcaag ttactgtgag tgaatcaaaa 120
agcctgatca ttaactgcac atactctgca actagtatcg gctaccccaa cctgttctgg 180
tacgtcaggt atcctggcga ggggctccag cttctgctga aagtgatcac cgcaggccag 240
aaagggtcta gtcgcggctt cgaggctact tacaacaagg aagcaaccag ttttcacctc 300
cagaaagcca gcgtccaaga atccgatagc gccgtgtact attgcgccct ctggagtggg 360
tcatggcagc ttatcttcgg atctggcacc cagctgaccg tgatgccaga cattcagaac 420
cccgagcctg ccgtgtatca gctgaaggac ccccggtcac aagatagcac cctgtgcctg 480
ttcaccgact ttgattccca gattaatgtg cctaagacaa tggaatctgg caccttcatc 540
acggacaaaa ccgtcctgga tatgaaggct atggacagca aatccaacgg tgccatagct 600
tggtccaatc agacatcttt cacttgccaa gacatcttca aggagaccaa tgccacctac 660
ccatcaagcg acgtgccgtg tgatgctact ctgacggaga agagcttcga gaccgacatg 720
aacctgaatt ttcagaacct gagcgtgatg ggcctgagaa tcctgctgct gaaggtcgcc 780
gggttcaatc tgctcatgac actgcggctg tggtcctct 819
<210> 2
<211> 912
<212> DNA
<213>Artificial sequence ()
<400> 2
atggccactc gactgctgtg ctacaccgtg ctgtgccttt tgggcgctag aatcctgaac 60
agcaaagtga tccagacccc tagatacctg gtcaagggcc aggggcaaaa agccaagatg 120
agatgcatcc cagagaaagg gcacccggtg gtgttctggt atcagcaaaa caaaaacaac 180
gagttcaagt ttctgattaa cttccagaac caagaagtgc tccagcaaat cgacatgaca 240
gagaagaggt tctcagccga atgcccctca aatagccctt gtagcctgga gatccagagc 300
tccgaagctg gagacagcgc cctgtacctg tgcgctagtc gcgattcacc agagcagtat 360
tttggaccag gcacccggct gaccgtgctg gaggatctgc gtaacgtgac tccccctaaa 420
gtctcactgt tcgagcccag caaggcagaa attgccaaca agcagaaggc caccctggtg 480
tgccttgccc ggggcttctt tcctgaccac gtcgagctga gttggtgggt taacggaaaa 540
gaagtccata gcggcgtgag caccgacccc caggcataca aggagtctaa ttacagttat 600
tgcctgagca gccggctgcg ggtgagcgcc accttctggc acaaccctag gaatcatttc 660
cgctgtcagg tccaatttca cggcctgtcc gaggaagaca aatggccaga ggggtctcca 720
aagccggtga ctcagaacat cagcgccgag gcctggggtc gtgctgattg tgggattacg 780
tccgcatctt atcatcaggg cgtgctgagc gccaccatcc tgtacgagat tctgctcgga 840
aaggccaccc tgtatgccgt gctggtgagc ggtctggtgc tgatggctat ggtgaagaag 900
aagaacagct ga 912
<210> 3
<211> 462
<212> DNA
<213>Artificial sequence ()
<400> 3
atgtacagga tgcagctgct cagctgcatc gccctgagcc tggccctggt cacaaacagc 60
gcccccacca gcagcagcac caagaagacc cagctccagc tggagcacct gctgctggac 120
ctccagatga tcctgaacgg catcaacaat tacaagaatc ccaagctgac tcgcatgctg 180
accttcaagt tttatatgcc taagaaagct accgagctga agcacctcca gtgcctggag 240
gaagagctga agccactgga agaggtgctg aacctggcac aatctaagaa tttccacctg 300
cggccccggg acctgatcag caacatcaat gtgatcgtgc tggagctgaa ggggtccgag 360
accaccttca tgtgcgaata tgcagatgag actgccacga ttgtggagtt cctgaaccgg 420
tggatcacat tttgtcagag tatcatttca accctgacat ga 462
<210> 4
<211> 81
<212> DNA
<213>Artificial sequence ()
<400> 4
agggctaaac gctccgggtc tggagcaaca aacttctctc tgctgaagca ggctggcgat 60
gtggaggaaa atcctgggcc a 81
Claims (10)
1.T cells, it includes coding specific recognition NY-ESO-1 TCR nucleic acid and expresses the specific recognition NY-
ESO-1 TCR, the nucleic acid include the second of the first nucleic acid fragment for encoding the α chains of the TCR and coding TCR β chains
Nucleic acid fragment, wherein, it is SEQ ID NO that first nucleic acid fragment, which has sequence,：1 nucleotide sequence, second nucleic acid
It is SEQ ID NO that fragment, which has sequence,：2 nucleotide sequence.
2. the T cell of claim 1, it also includes the nucleic acid of encoding Interleukin 2 and expresses interleukin 2, it is preferred that
It is SEQ ID NO that the nucleic acid of the encoding Interleukin 2, which has sequence,：3 nucleotide sequence.
3. the T cell of claim 2, wherein having joint between first nucleic acid fragment and the second nucleic acid fragment, it is preferred that
The joint is coding T2A nucleotide sequence.
4. the T cell of claim 3, wherein the TCR of the coding specific recognition NY-ESO-1 nucleic acid and coding leucocyte
There is joint, it is preferred that the joint is coding T2A nucleotide sequence between the nucleic acid of interleukin 2.
5. any one of claim 1-4 T cell, it is CD4+T cells or CD8+T cells, preferably CD8+T cells.
6. recombinant expression carrier, it includes coding specific recognition NY-ESO-1 TCR nucleic acid,
The nucleic acid includes the first nucleic acid fragment for encoding the α chains of the TCR, and it is SEQ ID NO that it, which has sequence,：1 nucleosides
Acid sequence,
The nucleic acid includes the second nucleic acid fragment for encoding the β chains of the TCR, and it is SEQ ID NO that it, which has sequence,：2 nucleosides
Acid sequence,
The carrier is the carrier of the expressing protein in mammal.
7. the recombinant expression carrier of claim 6, it also includes encoding Interleukin 2, i.e. IL-2 nucleic acid, it is preferred that institute
It is SEQ ID NO to state the nucleic acid of encoding Interleukin 2 to have sequence：3 nucleotide sequence.
8. the recombinant expression carrier of claim 6, it is viral vector, such as rhabdovirus expression vector, adenovirus vector are inverse
Transcription vector, herpesvirus vector or slow virus carrier, preferably slow virus carrier.
9. pharmaceutical composition, it contains any one of any one of claim 1-5 T cell or claim 6-8 restructuring
Expression vector, it is preferred that described pharmaceutical composition is used to treat or prevent cancer.
10. any one of claim 1-5 T cell or any one of claim 6-8 recombinant expression carrier are for making
Purposes in the standby medicine for treating or preventing cancer, it is preferred that wherein described cancer is selected from melanoma, breast cancer, lung cancer, forefront
Gland cancer, thyroid cancer, oophoroma, osteosarcoma and synovial cell sarcom, for example, osteosarcoma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557338A (en) * | 2017-09-19 | 2018-01-09 | 深圳市北科生物科技有限公司 | Specific recognition NY ESO 1 T cell and its united application with cell factor |
| CN108103106A (en) * | 2018-01-09 | 2018-06-01 | 河南省华隆生物技术有限公司 | A kind of pFTM3GW recombinant vectors and its preparation method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107557338A (en) * | 2017-09-19 | 2018-01-09 | 深圳市北科生物科技有限公司 | Specific recognition NY ESO 1 T cell and its united application with cell factor |
| CN108103106A (en) * | 2018-01-09 | 2018-06-01 | 河南省华隆生物技术有限公司 | A kind of pFTM3GW recombinant vectors and its preparation method and application |
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